The present invention relates to methods and products for producing increased numbers of hematopoietic cells, of restoring to preselected normal levels numbers of hematopoietic cells, to therapies for treating deficiencies in hematopoietic cells and to in vitro methodologies for culturing hematopoietic cells.
PT-100 is a dipeptide consisting of valine-prolineboronic acid (ValboroPro) designed to interact with the cell surface receptor CD26. CD26, a type II transmembrane protein is expressed on the cell surface of a number of cell types, including lymphocytes (Marguet, D. et al., Advances in Neuroimmunol. 3:209-215 (1993)), hematopoietic cells (Vivier, I. et al., J. Immunol. 147:447-454 (1991); Bristol, et al., J. Immunol. 149:367 (1992)) thymocytes (Dang, N. H. et al., J. Immunol. 147:2825-2832 (1991), Tanaka, T. et al., J. Immunol. 149:481-486 (1992), Darmoul, D. et al., J Biol. Chem. 267:4824-4833 (1992)), intestinal brush border membrane and endothelial cells. Cell surface associated CD26 is a sialoglycoprotein, with most of its mass on the outside of the cell.
CD26 has been best characterized on peripheral T cells where it functions as a potent costimulatory signal for T cell activation. Its surface expression is upregulated upon T cell activation (Dong, R. P. et al., Cell 9:153-162 (1996), Torimoto, Y. et al., J. Immunol 147:2514 (1991), Mittrucker, H-W. et al., Eur. J Immun. 25:295-297 (1995), Hafler, D. A. et al, J. Immunol. 142:2590-2596 (1989), Dang, N. H. et al., J. Immunol 144:409 (1990)). CD26 has also been identified in rodents as an important regulatory surface receptor in hematopoiesis and lymphoid development (Vivier, I. et al., J. Immunol. 147:447-454 (1991)). The primary structure of CD26 is highly conserved between species (Ogata, S. et al., J. Biol. Chem. 264:3596-3601 (1998)). In humans CD26 seems to be involved in the regulation of thymocyte activation, differentiation and maturation (Dang, N. H. et al., J. Immunol. 147:2825-2832 (1991); Kameoka, J. et al., Blood 85:1132-1137 (1995)). We have evidence that CD26 is expressed within the human and murine hematopoietic systems.
CD26 is an ectoenzyme with activity identical to that of Dipeptidyl Peptidase IV (DPP-IV), a serine type exopeptidase with high substrate specificity. It cleaves N-terminal dipeptides from proteins if the penultimate amino acid is proline, or in some cases alaninie (Fleischer, B. Immunol. Today 15:180 (1994)). PT-100 is a potent inhibitor of DPP-IV activity.
The prior art PCT published application WO94/03055 teaches methods of producing increased numbers of hematopoietic cells by administering inhibitors of DPP-IV. The teaching of this published application, however, is that dosages of at least 1 mg/kg body weight are necessary to achieve such hematopoietic cell increases. This published application also teaches that inhibitors are administered to mammals which have an established deficiency of hematopoietic cells. The teaching also suggests that cytokines be administered in conjunction with the inhibitors to increase the production of hematopoietic cells in a subject.
The invention is based upon a variety of surprising and unexpected findings. It has been discovered, unexpectedly, that the agents useful according to the invention stimulate growth factor production by stromal cells. It also has been discovered, unexpectedly, that the agents useful according to the invention stimulate proliferation of primitive hematopoietic progenitor cells, but do not stimulate directly the differentiation or proliferation of committed progenitor cells. It further has been discovered, unexpectedly, that the agents useful according to the invention can be administered at doses much lower than would have been expected according to the teachings of the prior art. Another unexpected finding is that the agents according to the invention can accelerate the time it takes to achieve hematopoietic cell recovery after treatment with an hematopoietic cell inhibitor. Another unexpected finding is that the agents useful according to the invention can at relatively low doses, restore normal levels of neutrophils at least as fast as the most successful commercially available product used worldwide for this purpose, except that the agents useful according to the invention can be used orally, whereas the commercially available product (which represents more than a billion dollar market) must be injected. These unexpected results have important therapeutic and experimental research implications.
According to one aspect of the invention, a method is provided for treating a subject to stimulate hematopoiesis in the subject. The invention involves administering to a subject in need of such treatment an amount of an agent effective to increase the number of hematopoietic cells or mature blood cells in the subject, wherein the amount is less than 1 mg/kg body weight per day and wherein the agent is a compound of Formula I.
The agents useful according to the invention are compounds of Formula I: 
wherein m is an integer between 0 and 10, inclusive; A and A1 are L-amino acid residues (for glycine there is no such distinction) such that the A in each repeating bracketed unit can be a different amino acid residue; the C bonded to B is in the L-configuration; the bonds between A and N, A1 and C, and between A1 and N are peptide bonds; and each X, and X2 is, independently, a hydroxyl group or a group capable of being hydrolysed to a hydroxyl group in aqueous solution at physiological pH. By xe2x80x9cthe C bonded to B is in the L-configurationxe2x80x9d is meant that the absolute configuration of the C is like that of an L-amino acid.
Thus, the 
group has the same relationship to the C as the xe2x80x94COOH group of an L-amino acid has to its xcex1 carbon. In some embodiments, A and A1 are independently proline or alanine residues; m is 0; X1 and X2 are hydroxyl groups; the inhibitor is L-Ala-L-boroPro; and the inhibitor is L-Pro-L-boroPro.
In one important aspect of the invention, the subject has an abnormally low level of hematopoietic cells or mature blood cells and the agent is administered in an amount effective to restore levels of a hematopoietic cell-type or mature blood cell-type to a preselected normal or protective level. The agent preferably is administered to the subject in at least 2 doses in an 18 hours period. The invention has particularly important applications in the restoration of normal or protective levels of neutrophils, erythrocytes and platelets. The most preferred agent is ValBoroPro.
According to another aspect of the invention, a method is provided for shortening or eliminating the time that a subject has an abnormally low level of hematopoietic or mature blood cells resulting from treatment with a hematopoietic cell inhibitor. An agent is administered to a subject in need of such treatment in an amount effective to increase the number of hematopoietic cells or mature blood cells in the subject, wherein the administration of the agent begins prior to or substantially simultaneous with administration of the hematopoietic cell inhibitor. The agents and the preferred agent are as described above. In one important embodiment, the hematopoietic cell inhibitor causes an abnormally low level of hematopoietic cells or mature blood cells in the subject and the agent is administered in an amount effective to restore levels of a hematopoietic cell type to a preselected normal or protective level. Preferably, the agent is administered to the subject in at least 2 doses in an 18 hour period. In important embodiments, the agent is used to restore in the subject normal or protective levels of neutrophils, erythrocytes or platelets. The preferred effective amount of agent is as described above.
According to another aspect of the invention, a method is provided for preparing a subject for treatment with a hematopoietic cell inhibitor. The method involves administering to the subject prior to the subject receiving the hematopoietic cell inhibitor an agent in an amount effective to stimulate in the subject production of growth factors. In one embodiment the agent stimulates stromal cell production of growth factor. The agents and the preferred agent are as described above. In one important embodiment, the growth factor is granulocyte colony stimulating factor. In other embodiments the growth factor is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17, TPO, EPO, MCSF, GMCSF, FLT-3 Ligand and Stem Cell Factor. Preferably, the amount administered to the subject is less than 1 mg/kg body weight per day. It also is preferred that the administration of the agent be in at least 2 doses of the agent in an 18 hour period.
According to another aspect of the invention, a method is provided for treating a subject to increase the number of hematopoietic cells or mature blood cells in the subject. An agent is administered to a subject in need of such treatment in an amount effective to increase hematopoietic cells or mature blood cells in the subject, wherein the agent is administered in a first regimen consisting of 2 doses or 3 doses in an 18 hour period. The agents and the preferred agent are as described above. In one important embodiment, the agent is administered in a second regimen consisting of 2 doses or 3 doses in an 18 hours period, wherein the second regimen is separate in time from the first regimen. In another embodiment, the agent is administered in a third regimen consisting of 2 doses or 3 doses in an 18 hour period, wherein the third regimen is separate in time from the first and second regimens. In other embodiments, the agent is administered optionally in a fourth regimen, a fifth regimen, a sixth regimen, or a seventh regimen, wherein each of such regimens consists of 2 doses or 3 doses in an 18 hours period, and wherein the regimens are separate in time from one another and from the prior regimens. In one important embodiment, the subject has an abnormally low neutrophil count and the amount is effective to restore in the subject a preselected level of neutrophils. In other important embodiments the subject has abnormally low levels of erythrocytes and platelets. The preferred dosages, agents, and the like are as described above. In important embodiments, the dosage is no more than six regimens, no more than five regimens, no more than four regimens, no more than three regimens, and even no more than two regimens.
According to another aspect of the invention, a method is provided for preparing a subject""s cells for reintroduction into the subject. The method involves treating the subject with an agent in an amount effective to stimulate in the subject the hematopoietic cells, then collecting the hematopoietic cells from the subject. The collected cells later are reintroduced into the subject. The collected cells optionally can be ex vivo cultured. The agents and preferred agent are as described above. In one embodiment, the ex vivo culturing is carried out in the presence of an amount of the agent effective to stimulate proliferation of the collected cells. In another embodiment, the concentration of the agent in medium surrounding the collected cells is less than 10xe2x88x928 moles per liter, and less than 10xe2x88x929 moles per liter and even less than 10xe2x88x9210 moles per liter.
According to another aspect of the invention, a method is provided for stimulating growth factor production by stromal cells. The method involves contacting the stromal cells with an agent in an amount effective to stimulate growth factor production by the stromal cells. The agents and the preferred agent are as described above. In one embodiment, the stromal cells are in an in vitro layer of stromal cells for supporting early progenitor cell growth and further comprising culturing the stem cells in the presence of these stromal cells. In another embodiment, the stromal cells are in vivo in a subject. In another embodiment, the growth factor is granulocyte colony stimulating factor. In other embodiments the growth factor is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17, TPO, EPO, MCSF, GMCSF, FLT-3 Ligand and Stem Cell Factor. In an in vivo embodiment, the agent is administered to a subject in an amount less than 1 mg/kg body weight per day. In still another embodiment, the stem cells are cultured in an environment free of exogenously added granulocyte colony stimulating factor. In important embodiments the stromal cells are bone marrow or thymic stromal cells.
According to another aspect of the invention, a kit is provided for treating a subject having an abnormally low level of hematopoietic cells resulting from treatment with a hematopoietic cell inhibitor or for treating prophylactically a subject being treated with a hematopoietic cell inhibitor to prevent decrease or loss of hematopoietic and/or mature blood cells. The kit is a package containing a first dosage and instructions for treating a subject substantially simultaneous with or prior to treatment with the hematopoietic cell inhibitor. The package also contains a second dosage and instructions for treating a subject only after treatment with the hematopoietic cell inhibitor. The dosages are in effective amounts and the agents and preferred agent are as described above. In one embodiment, the second dosage is between 2 and 5 regimens, each of the regimens consisting of 2 or 3 doses per day of the agent. In one embodiment, the combination of the doses is less than 1 mg/kg body weight per day. One preferred kit is for treatment of neutropenia. Other preferred kits are for treatment of an abnormally low level of erythrocytes or platelets.
According to still another aspect of the invention, a kit is provided for treating a subject having abnormally low level of hematopoietic cells. The kit is a package containing a complete dosage for restoring normal levels of a hematopoietic cell type. The package consists essentially of: (1) a first dosage in an effective amount for administration to the subject during a first day, (2) a second dosage in an effective amount for administration to the subject during a second day, (3) optionally, a third dosage in an effective amount for administration to the subject during a third day, (4) optionally, a fourth dosage in an effective a mount for administration to the subject during a fourth day, (5) optionally, a fifth dosage in an effective amount for administration to the subject during a fifth day, (6) optionally, a sixth dosage in an effective amount for administration to the subject during a sixth day and (7) optionally, a seventh dosage in an effective amount for administration to the subject during a seventh day. The agents and preferred agent are as described above. In one important embodiment, each of the dosages consists of 2 or 3 doses of the agent for administration each day. Preferred doses and dosages are as described above. In important embodiments, the kit consists essentially of less than 5, less than 4, and less than 3 and even less than 2 dosages.
These and other aspects of the invention will be described in greater detail below.